Fisheries Science 65(4), 613-617 (1999)

Purification and Characterization of L-Methionine Decarboxylase from Crypthecodinium cohnii

Hirotaka Kitaguchi,*1 Aritsune Uchida,*' and Yuzaburo Ishida*2 *'Division of Applied Bioscience , Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan *2Department of Marine Biotechnology , Faculty of Engineering, Fukuyama University, Fukuyama, Hiroshima 729-0292, Japan

(Received January 5, 1999)

L-Methionine decarboxylase [EC 4.1.1.571 was purified from the marine dinoflagellate Cryp thecodinium cohnii. After four purification steps including anion exchange chromatography and size exclusion chromatography, the enzyme was purified 215-fold and the yield was 0.1%. The purified en zyme showed a single protein band on polyacrylamide gel electrophoresis. The molecular weight of the enzyme estimated by gel filtration was 204,000. The optimum pH and temperature of the enzyme activi ty were 7.3 and 30°C, respectively. The enzyme activity was stimulated by the addition of pyridoxal phosphate (PLP) and was inhibited by the typical inhibitors of PLP dependent enzymes. The decarbox ylated product of L-methionine by the enzyme was 3-methylthiopropanamine (MTPA). The enzyme should be the key enzyme in dimethylsulfoniopropionate (DMSP) biosynthesis from L-methionine in C. cohnii. This shows that C. cohnii has the DMSP biosynthetic pathway from L-methionine to DMSP via MTPA.

Key words: L-methionine decarboxylase, purification, Crypthecodinium cohnii, dinoflagellate, dimethylsulfoniopropionate, 3-methylthiopropanamine

Dimethylsulfoniopropionate (DMSP) is the precursor r zation of the enzyme. of dimethylsulfide (DMS), the major sulfur compound released from ocean to the atmosphere. As over 90% of Materials and Methods sulfur emitted from ocean is DMS, DMS has significance for the global sulfur cycle.') Emitted DMS plays an im Culture portant role in the formation of cloud condensation C. cohnii (ATCCe32001), which was able to grow heter nuclei,2,3)thereby it may commit to the acid precipitation otrophically, was cultured in an ESW medium (2% glu and the climate regulation. The main DMSP producers are cose and 0.2% yeast extract (DIFCO) in filtered sea water) marine macroalgae,4-7) and microalgae.8-10) The possible at 25°C in dark condition. Four days culture was harvest biological function of DMSP is an osmolyte in such ma ed by centrifugation and used for further experiments. rine algae.11)All known osmolytes have the same character istics as "compatible solute", that is defined as the high Crude Extract Preparation solubility to the water and the inaffectivity to the cell C. cohnii cells were homogenized with 10 volumes of metabolism. DMSP was accumulated in relation to the ex cold acetone (-20•Ž) and the solid residue was collected ternal osmolarity in macroalgae5,12,13)and microalgae,9,14,15) by Buchner filtration and then lyophilized. The acetone and DMSP had no effect to the enzymes isolated from the powder was sonicated in 20 volumes of buffer A (50 mm DMSP producing microalgae.l6)These data suggests that potassium phosphate buffer, pH 7.2) for 10 min at 0•Ž by DMSP has a role of "compatible solute" in some algae. It using UR-200P ultrasonic disrupter (TOMY Seiko Co. has also been suggested that DMSP is a cryoprotectant in Ltd.). The suspension was centrifuged at 25,000 •~ g for 30 polar species.") min and the pellet was discarded. The biosynthetic pathway of DMSP has been little known. In a higher plant Wollastonia biflora, DMSP is Ammonium Sulfate Precipitation derived from S-methylmethionine,18.19) while DMSP is der Ammonium sulfate was added into the supernatant to ived from L-methionine in a green macroalga Enteromor 80% saturation and stirred over 30 min. Following cen pha intestinalis.20) We previously reported that L-methio trifugation at 25,000 •~ g for 30 min, the pellet was dis nine was the close precursor of DMSP in a marine solved in buffer A and dialyzed against 100 volumes of dinoflagellate Crypthecodinium cohnii,21) which produce a buffer A twice. large amount of DMSP. The radiotracer experiment showed the first step of the synthetic pathway was decar DEAE-cellulose Column Chromatography boxylation of L-methionine.21) As we found the L-methio The sample was centrifuged at 20,000 •~ g for 20 min to nine decarboxylase activity in the crude extract of C. remove particles interfering with column chromato cohnii, we report here the purification and some characteri graphy. Then the sample was loaded to a DEAF-cellulose 614 Kitaguchi et al .

DE52 (Whatman Co.) column (5 x 20 cm) equilibrated Enzyme Assay with buffer A. Proteins were eluted with a linear gradient L-Methionine decarboxylase assay was based on meas of 0 to 0.6 M NaCl in buffer A at a flow rate of 1.5 ml/ urement of the release of 14C02 from L-[l-14C]-methio min. The total elution volume was 400 ml . nine. The reaction mixture contained 20 mm of L-methio nine (with 37 kBq of L-[l-14C]-methionine (2.0 GBq/ Resource-Q Column Chromatoera mmol, American Radiolabeled Chemicals Inc.)), 1 mm of The active fractions from the DEAE-cellulose column pyridoxal 5•Œ-phosphate (PLP), and the enzyme in a total were pooled and added ammonium sulfate to 80% satura volume of 1 ml of buffer A in a test tube with butyl cap tion. The precipitate was dissolved in buffer A and dia ping. After incubation at 30•Ž for 10 min, the reaction lyzed. Then the sample was loaded to a Resource-Q (Phar mixture was acidified with 0.1 ml of 50% trichloroacetic macia) column equilibrated with buffer A . Proteins were acid (TCA) to stop reaction and to volatilize dissolved eluted with a linear gradient of 0 to 0.5 M NaCl in buffer A CO2. The head space gas was bubbled into a trap solution at a flow rate of 2 ml/min. The total elution volume was (1 ml of 2-aminoethanol, 1.5 ml of 2-methoxyethanol, and 25 ml. 7.5 ml of Aquasol-2 (NEN Life Science Products Inc.)). Trapped 14CO2 was determined by using a liquid scintilla Mono-Q Column Chromatography tion counter (Aloka LCS-3050). One unit of enzyme activi The active fractions from the Resource-Q column were ty was defined as the amount which released l ƒÊmol of pooled and added ammonium sulfate to 80% saturation. CO2 per I min under above conditions. The precipitate was dissolved in buffer A containing 0 .2 M NaCland dialyzed against the same buffer. Then the sam Detection of L-methionine Decarboxylase Product ple was loaded to a Mono-Q (Pharmacia) column The L-methionine decarboxylase product was deter equilibrated with buffer A containing 0.2 M NaCl. Proteins mined by two-dimensional thin layer chromatography were eluted with a linear gradient of 0.2 to 0.4 M NaClin (TLC). Reaction mixture (1 ml) containing 20 mm of L buffer A at a flow rate of 0.5 ml/ min. The total elution - methionine with 370 kBq of L-[35S]-methionine (40 TBq/ volume was 20 ml. mmol, American Radiolabeled Chemicals Inc.), 1 mM of PLP, and 1,ƒÊg of the purified enzyme was incubated for 1 Superdex 200HR Column Chromatography h at 30•Ž. The reaction mixture was added 0.1 ml of 50% The active fractions from the Mono-Q column were col TCA and then centrifuged. The supernatant was neutral lected and dialyzed against buffer B (0.2M potassium phos ized with KOH and spotted onto a DC-Fertigplatten cellu phate buffer, pH 7.2). Then the sample was concentrated lose TLC plate (Merck). Two solvents (1-butanol:acetic by Ultrafree-MC (Millipore) centrifugal filtration to the acid:water=4:1:1, phenol:water=7:3) were used for de volume of 200ƒÊ1 and loaded onto a Superdex 200HR velopment. The radiolabeled compounds were detected by (Pharmacia) column equilibrated with buffer B. Gel filtra the bioimaging analyzer BAS-2000 (Fuji Film Co. Ltd.). tion was performed at a flow rate of 0.5 in// min. For deter mination of molecular weight, the column was calibrated Results with reference proteins (blue dextran, 2,000,000; thyroglobulin, 669,000; ferritin, 440,000; catalase, Purification of L-methionine Decarboxylase 232,000; aldolase, 158,000; bovine serum albumin (BSA), The purification of L-methionine decarboxylase is sum 67,000). marized in Table 1. The enzyme was purified 215-fold with a final specific activity of 4.45 unit/mg under the standard Electrophoresis assay condition and with the overall yield of 0.17%. Polyacrylamide gel electrophoresis (PAGE) was carried The sample obtained from gel filtration on a Superdex out according to the Laemmli's method,22) using a 7.5% 200HR column gave a single band on both native PAGE resolving gel and a 4% stacking gel with or without SDS. (Fig. IA) and SDS-PAGE. The band position on native Proteins in gels were detected with Silver Stain Plus (Bio- PAGE corresponded to that of active fractions of gel Rad Labs.). sliced assay (Fig. IB).

Protein Assay Molecular Properties of L-methionine Decarboxylase Protein quantity was measured by the method of Brad Molecular weight estimated by gel filtration was 204,000 ford") with a Bio-Rad Protein Assay (Bio-Rad Labs.) and for the native enzyme (Fig.2A). SDS-PAGE of the active BSA as a standard. fractions of Superdex 200HR showed a single band with a molecular weight of approximately 100,000 (Fig. 2B).

Table 1. Summary of purification of L-methionine decarboxylase from Crypthecodinium cohnii Purification of L-methionine Decarboxylase 615

Fig. 1. A: Native polyacrylamide gel electrogram of L-methionine decarboxylase purified from C. cohnii. The protein was stained with Silver Stain Plus (Bio-Rad Labs.). B: Gel sliced assay of L-methionine decarboxylase. After purified en zyme was loaded onto native PAGE, the gel was sliced every 2 mm . The sliced gels were soaked into buffer A and the activities were meas ured.

These results suggest that the enzyme is 204,000 homodimer consisting of 100,000 subunits.

Effects of pH and Temperature The activity of the purified L-methionine decarboxylase was determined in potassium citrate-NaOH buffer (pH 4 6), phosphate buffer (pH 6-8), and Tris-HCl buffer (pH Fig. 2. Molecular weight determination of L-methionine decarboxy 7.7-9). The optimum pH for the enzyme activity was pH lase. 7.3 (Fig. 3). L-Methionine decarboxylase activity was deter A: Determined by Superdex 200HR gel filtration. 1, Thyroglobu lin (Mr; 669,000); 2, ferritin (Mr; 440,000); 3, catalase (Mr; 232,000); mined at a temperature range of 15-55•Ž. The optimum 4, L-methionine decarboxylase; 5, aldolase (Mr; 158,000); 6, BSA temperature for the enzyme activity was 30•Ž (Fig. 4). (Mr; 67,000). Kav=(Ve-Vo)/(Vt-Vo); Vo: void volume, Ve: elu tion volume, Vt: bed volume. B: Determined by SDS-PAGE. 1, Myo Effect of Coenzyme sin (Mr; 212,000); 2, ƒ¿-macroglobulin (Mr; 170,000); 3, ƒÀ-galactosi PLP is a major coenzyme for decarboxylases. The PLP dase (Mr; 116,000); 4, L-methionine decarboxylase; 5, transferrin requirement for L-methionine decarboxylase from C . (Mr; 76,000); 6, glutamic acid dehydrogenase (Mr; 53,000). cohnii was determined. Enzyme assay was performed without or with 1,ƒÊM to 1 mm PLP. The activity depended on the concentration of PLP (Table 2). The typical inhibi fore, the acetone precipitation was carried out for remov tors of PLP enzymes (phenylhydrazine, semicarbazide ing the oil and it was able to store the activity for several hydrochloride, carboxymethoxylamine hemihydrochlo months. During the purification, the low recovery of the ride, and hydroxylamine) inhibited the L-methionine enzyme was a problem. One of the reasons was the instabil decarboxylase activity (Table 3). ity of the enzyme, but it had little effect for stabilizing the enzyme to add PLP, 2-mercaptoethanol, or glycerol. Un Product from L-methionine by L-methionine Decarboxylase like Dryopteris filix-mas L-methionine decarboxylase,241 C. cohnii L-methionine decarboxylase was not membrane The product from L-methionine by purified L-methio bound because we were able to solubilize the activity into nine decarboxylase was analyzed by two-dimensional the buffer solution without detergents. TLC. The product was developed to the same position of C. cohnii L-methionine decarboxylase had the same the authentic 3-methylthiopropanamine (MTPA) (Fig. 5). homodimeric structure as all other reported L-methionine decarboxylases,24-2) but the molecular weight was about Discussion two times larger than other ones. The pH optimum of C. cohnii L-methionine decarboxylase (pH 7.3) was similar to C. cohnii contained a large amount of oil and it inter Streptomyces (pH 6.9)25)but different to D. filix-mas (pH fered with the purification procedure of the enzyme. There 5.0).24) 616 Kitaguchi et al.

Fig. 5. Two dimensional TLC analysis of labeled sulfur compounds in the products of L-methionine decarboxylase from L-[35S]-methio nine. The reaction mixtures were incubated for 1 h. A: With the en zyme, B: Without the enzyme. 1: MTPA, 2: L-methionine, 3: Methionine sulfoxide.

Fig. 3. Effect of pH on L-methionine decarboxylase activity. The activity was determined with following buffers (final concen tration of 50 mm); •¡ potassium citrate-NaOH buffer, •œpotassium

phosphate buffer, •£ Tris-HCl buffer.

Fig. 4. Effect of temperature on L-methionine decarboxylase activity.

Table 2. Effect of PLP concentration on L-methionine decarboxy lase activity

Fig. 6. Possible pathway from L-methionine to DMSP in C. cohnii. MTPA: 3-Methylthiopropanamine, MTP: 3-Methylthiopro pionate, DMSP: Dimethylsulfoniopropionate.

Amino acid decarboxylases are classified in two groups. One is the PLP-dependent enzyme that requires PLP as a coenzyme and the other is the pyruvoyl enzyme that con Table 3. Effect of inhibitors on PLP dependent enzymes tains a pyruvoyl residue in the active site. All of the previously reported L-methionine decarboxylases are PLP-dependent enzymes .24-26)C. cohnii L-methionine decarboxylase was significantly stimulated by the addition of PLP. The retained PLP in the enzyme might be the rea son for the remaining activity without adding PLP (Table 2). The activity was inhibited by typical inhibitors of PLP - dependent enzymes. These results strongly suggest that C. cohnii L-methionine decarboxylase is a PLP-dependent en zyme. Purification of L-methionine Decarboxylase 617

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